Q-PCR

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OUTLINE

 What is PCR and purpose of it?

    What is Q-PCR and purpose of it?

How does Q-PCR work?

Types of Q-PCR probes and comparison of types Advantages and Disadvantages of Q-PCR vs. PCR  Questions

What is PCR?

   Stands for Polymerase Chain Reaction #copies of DNA fragment(X)=X o (1+E) n where n=#of cycles in PCR reaction and E=efficiency Steps in PCR    Denaturation(95 o C)~1min Annealing(55 o C)~45sec Extension(72 o C)~2min  Cycle thorugh 25-35

Purpose of PCR

   Easy to sequence some of million copies and detect rather than trying to sequence and detect a single copy of a gene Can calculate which sample is biggest when comparing two or more DNA fragments It is used to clone specific genes

What is Q-PCR?

     Stands for Quantitative Polymerase Chain Reaction Assay that monitors accumulation of DNA from a PCR reaction Important technique to quantify RNA(mRNA) levels and DNA gene levels in biological samples Templates : DNA, cDNA,RNA Similar to PCR except the progress is monitored by a camera or detector  Uses fluorescence-based probes to detect DNA or RNA  Data collection start at early exponential phase and examined at the same time with detection

Research Objectives

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Gene validation

  Primary validation Confirmation of microarray data 

Viral detection

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Bacterial detection and identification

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Gene duplication or DNA quantification

www.scienceboard.net

Applications

 Pathogen detection      GMO analysis Quality control Forensics Methylation studies Detect proteins with Q-PCR    DNA/RNA quantification Protein stability testing Drug therapy efficacy / drug monitoring

Q-PCR

  Assay uses a standard curve to quantitate the amount of target present using a fluorescence labeled probe for detection. Each technique uses some kind of fluorescent marker which binds to the DNA

Types of Q-PCR

   Hydrolyzation based Assays  Taqman, Beacons, Scorpions DNA-binding agents  SYBR Green Hybridization based Assay  Light cycler(Roche)

Taqman Probes

   Fluorescence-labeled oligonucleotides (TaqMan® probes) TaqMan probes are complementary to a region of the target gene The 5' to 3' exonuclease activity of the polymerase cleaves the probe, releasing the fluorophore into solution

Characteristics of Taqman Probes

    Oligonucleotides longer than the primers (20-30 bases long with a Tm value of 10 oC higher) that contain a fluorescent dye usually on the 5' base, and a quenching dye typically on the 3' base The excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing(FRET) Uses universal thermal cycling parameters and PCR reaction conditions One specific requirement for fluorogenic probes is that there be no G at the 5' end

Molecular Beacons

  Contain fluorescent and quenching dyes at either end but they are designed to adopt a hairpin structure while free in solution to bring the fluorescent dye and the quencher in close proximity for FRET to occur Have two arms with complementary sequences that form a very stable hybrid or stem

molecular beacons

Excitation FRET

A B C Reporter Non-fluorescent Quencher

ANNEALING Amplicon

SYBR Green I

   A fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA Binds to the minor groove of the DNA double helix with a higher affinity for dsDNA than for single-stranded DNA (ssDNA) Fluorescence is greatly enhanced (1000-fold) upon DNA-binding making this dye a sensitive indicator for the quantity of dsDNA

 http://www.youtube.com/watch?v=5ZEySHfCWAU&feature=related

Taqman vs. SYBR Green I

TaqMan Probe

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SYBR Green Advantages:

 Increased specificity   Use when the most accurate quantitation of PCR product accumulation is desired.

Option of detecting multiple genes in the same well (multiplexing).

Disadvantages:

 Relative high cost of labeled probe.

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Advantages:

Relative low cost of primers.

No fluorescent-labeled probes required.

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Disadvantages:

Less specific – only primers determine specificity.

Specific and non-specific double-stranded PCR products generate the same fluorescence signal upon binding SYBR Green I dye.

Not possible to multiplex multiple gene targets.

Q-PCR vs. PCR

Some of the problems with End-Point Detection:          Poor Precision Low sensitivity Short dynamic range < 2 logs Low resolution Non - Automated Size-based discrimination only Results are not expressed as numbers Ethidium bromide for staining is not very quantitative Post PCR processing

Eventually the reactions begin to slow down and stop all together or plateau.Each tube or reaction will plateau at a different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase.

The plateau phase is where traditional PCR takes its measurement, also known as end-point detection .

 Hard to differentiate between the 5-fold change on the Agarose gel. Q-PCR is able detect a two-fold change (i.e. 10 Vs. 20 copies).

BioRad iCycler

References

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Dorak MT (Ed): Real-Time PCR (Advanced Methods Series). Oxford: Taylor & Francis, 2006 http://dorakmt.tripod.com/genetics/realtime.html

http://www.protocolonline.org/prot/Molecular_Biology/PCR/Real-time_PCR/index.html

SYBR Green Quantitative PCR Protocol

http://www.genetics.ucla.edu/labs/lusis/greenquantitative.htm

Quantification using real-time PCR technology< http://www.wzw.tum.de/gene quantification/klein-2002.pdf

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Real-Time PCR (qPCR) Basics

< http://www.primerdesign.co.uk/Download%20material/Beginners%20guide%20to%20real time%20PCR.pdf

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QUESTIONS?